School of Biology Seminar Series
School of Biology Seminar Series
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ItemMapping Protein Folding on Organismal Fitness One Mutation at a Time(Georgia Institute of Technology, 2013-03-12) Shakhnovich, EugeneIn this presentation I will describe our efforts at understanding how molecular properties of proteins determine fitness landscape of populations of carrier organisms. Recent multi-scale evolutionary models, which assume certain relationship between organismal fitness and stability of their proteins, have been successful in predicting such biological phenomena as lethal mutagenesis (six mutations per genome per generation), distributions of protein stabilities (‘’marginal’’ protein stability being a consequence of a mutation-selection balance), correlation between evolutionary rates and abundances. However, many of the underlying assumptions of these models have not been tested experimentally. Our recent efforts aim to close this gap. We explore fitness landscape of E.coli through controlled rational mutational genomic perturbations of expression level and stability of essential protein Dihydrofolate Reductase (DHFR). To that end we created transgenic E.coli, which carry specified mutations in the folA gene encoding DHFR and also placed the folA gene under an IPTG controllable promoter, making it possible to change the intracellular abundance of DHFR in a wide range. Using competition essays, we measured how biological fitness depends on Biophysical properties of DHFR such as its abundance in the cytoplasm, stability of its native state and folding intermediate, and catalytic activity. Mutant DHFR proteins in a few strains aggregated rendering them nonviable but the majority exhibited fitness higher than wild type at a growth temperature of 42oC. We found that mutational destabilization of DHFR proteins in E. coli is counterbalanced by soluble oligomerization that restores their structural stability and protects from aggregation. Further, we found that protein homeostasis plays a defining role in sculpting fitness effect of mutations. In particular, overexpression of GroEL as well as deletion of one of the proteases, Lon, resulted in complete recovery of fitness of unviable strains. Further study, including in vitro essays of ANS binding showed that GroEL and Lon compete for folding intermediate of DHFR and their relative concentrations determines the outcome. We developed a computational model to analyze this competition, which lead us to the conclusion that our observations cannot be reconciled with GroEL role as just caging device to protect DHFR mutants from aggregation and proteolysis. Rather, it must play an active role converting intermediate to folded molecules.
ItemGenetics under geothermal conditions: Homologous recombination in the archaeon Sulfolobus acidocaldarius(Georgia Institute of Technology, 2008-10-23) Grogan, DennisHyperthermophilic archaea differ radically from all model organisms with respect to their evolutionary history and the severe environmental conditions they require. This divergence raises questions as to whether their genetic processes also have unusual properties; but few of these processes have been analyzed in vivo. In the extreme thermoacidophile Sulfolobus acidocaldarius, a conjugational mechanism of DNA transfer enables recombination between chromosomal mutations to be quantified. Early studies of this system suggested a non-reciprocal mechanism in which donor sequences become incorporated into the recipient genome as short segments. Subsequent studies using electroporation found that synthetic oligonucleotides can recombine into this genome. When similar experiments used longer, duplex DNAs containing multiple, silent markers, the resulting recombinants often contained multiple replacement tracts, consistent with an unusual, "short-patch" mode of homologous recombination.
ItemSteroidogenesis-inducing protein: An enigmatic protein with multiple biological functions(Georgia Institute of Technology, 2008-10-09) Khan, Shafiq A.SIP was isolated and characterized from human ovarian follicular fluid in our laboratory on the basis of its profound effects on steroid production in testicular, ovarian and adrenal cells. Later studies showed that SIP is also a potent mitogen and stimulated DNA synthesis in testicular Leydig cells, ovarian granulosa cells and in cell lines derived from ovarian epithelial carcinomas. Partial amino acid sequence analysis of this protein revealed that SIP is a novel protein which shows similarities with immunoglobulins and with a recently characterized DING family of proteins. Antibodies raised against specific SIP peptide blocked the activity of SIP on DNA synthesis and on steroid production in testicular cells. Using these antibodies we also determined the expression of SIP in different tissues and cell lines including prostate cancer cells. A SIP protein was detected in the rat testes, ovarian granulosa cells, ovarian epithelial cancer cell lines and in several prostate cancer cell lines. Furthermore, treatment with purified SIP resulted in induction of proliferation of prostate cancer cells similar to that seen in ovarian cancer cells and in other cell types. Based on these studies we hypothesize that SIP is produced by prostate cancer cells in the advanced stages of disease and serves as an autocrine regulator of cell proliferation in these cells. Furthermore, we hypothesize that SIP may exert its steroidogenic effects on these cells resulting in synthesis of steroids which may serve as ligands for AR and hence may lead to insensitivity to exogenous androgens
ItemVisualizing Biological Machines with CryoEM(Georgia Institute of Technology, 2008-09-22) Carragher, BridgetOver the past decade, cryo-electron microscopy (cryoEM) has emerged as a powerful approach to the structural determination of large macromolecular complexes. Elucidating the structure and mechanism of action of these "molecular machines" is an emerging frontier in understanding how the information in the genome is transformed into cellular activities. In cryoEM the macro molecular specimen is preserved in a thin layer of vitreous (glassy) ice and imaged in the electron microscope using very low doses of electrons. The low signal to noise ratio of the resulting images means that averaging is required to recover the signal and reconstruct a three dimensional map of the structure. Our goal is to develop a pipeline to automate the processes involved in solving macromolecular structures using cryo-electron microscopy. One of the goals of the pipeline is to enable much higher data throughputs and improve the resolution of single particle reconstructions. We are also using the pipeline to help understand what currently limits resolution in these maps. The current status of these efforts will be illustrated using a variety of macromolecules as case studies.